Factors affecting the activation of rabbit muscle phosphofructokinase by actin Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/xw42nb941

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  • The consistent application of phosphatase inhibitors and a novel final purification step using a connected series of DE-51, DE-52, and DE-53 anion exchange chromatography columns facilitate the preparation of electrophoretically homogeneous sub-pupulations of rabbit muscle phosphofructokinase which differ in their catalytic properties and endogenous covalent phosphate contents. A band of "high" phosphate enzyme (fraction II) flanked by regions of "low" phosphate enzyme (I and III) is an unusual feature of the final purification profile. Fractions I (in this case 0.42 mol P/84,000 g enzyme) and II (1.26 mol P/84,000) exhibit the most pronounced functional differences of the fractions. Both are activated by the addition of rabbit skeletal muscle F-actin. Under the assay condition, K[subscript m] of phosphofructokinase activity occurs at 15.4 nM actin monomer for fraction I and 9.7 nM for fraction II. The "low" phosphate enzyme is synergistically activated in the presence of 0.12 μM actin plus 3.0 μM F 2,6-P₂, with a marked increase in V[subscript max], while the "high" phosphate enzyme is not. Neither fraction is activated appreciably by the addition of G-actin or the chymotrypsin-resistant actin "core". The covalently cross-linked trimer of actin stimulates the activity of both the "low" and "high" phosphate enzyme fractions. However, the previously mentioned synergistic activation characteristic of fraction I fails to occur in solutions containing the trimer plus F 2,6-P₂. In vitro phosphorylation of fraction I catalyzed by the cAMP-dependent protein kinase causes its properties to become more like, but not the same as those of fraction II. The total amount of covalent phosphate present approaches 2 mol P/84,000 g for both fractions. Alkaline phosphatase and calcineurin catalyze partial (30-45%) dephosphorylation of the enzyme. Pilot studies just initiated with the cGMP-dependent protein kinase show that it catalyzes the incorporation of 1.03 mol P/mol protomer at a rate 1.7 fold greater than obtained with the cAMP-dependent protein kinase. Two actin binding proteins, filamin and a-actinin, influence only the activity of "low" phosphate PFK-with resulting activation and inhibition, respectively--in the absence of actin. The addition of excess actin to the solutions largely reverses these effects.
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